Skip to content
1887

Journal of Medical Microbiology logo Journal of Medical Microbiology

Volume 54, Issue 1

Safe susceptibility testing of by flow cytometry with the fluorescent nucleic acid stain SYTO 16 No Access

Abstract

The time needed to obtain susceptibility results of using classical methodologies is still too long, and flow cytometry is a promising technique in the setting of the clinical laboratory, giving fast results. A safe, reliable and rapid method to study susceptibility to streptomycin, isoniazide, rifampicin and ethambutol is described. Isolates of mycobacteria, grown for 72 h in the absence or presence of antimycobacterial drugs in the mycobacteria growth indicator tube (MGIT), were heat-killed, stained with SYTO 16 (a nucleic acid fluorescent stain that only penetrates cells with severe lesion of the membrane) and then analysed by flow cytometry. Sixteen strains with different susceptibility patterns were tested and an excellent correlation with the BACTEC MGIT 960 protocol for susceptibility was shown. In contrast to resistant strains, sensitive strains lose their cellular integrity after incubation with antimycobacterial drugs, allowing SYTO 16 to penetrate the cells. Comparing the intensity of fluorescence of cells incubated with antimycobacterial drugs with that of drug-free cells, after staining with SYTO 16, it was possible to distinguish between sensitive, intermediate and resistant phenotypes. Other cytometric assays have been described for mycobacteria susceptibility testing but these have lower accuracy and safety. The described flow cytometric assay is a simple, fast, safe and accurate way to determine susceptibility of . .

  • Received:
  • Accepted:
  • Published Online:
© Microbiology Society
Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.45627-0
2005-01-01
2025-01-21
Loading full text...

Full text loading...

References

  1. Ardito F., Posteraro B., Sanguinetti M., Zanetti S., Fadda G. 2001; Evaluation of BACTEC Mycobacteria Growth Indicator Tube (MGIT 960) automated system for drug susceptibility testing of Mycobacterium tuberculosis . J Clin Microbiol 39:4440–4444 [CrossRef]
     [Google Scholar]
  2. Banaiee N., Bobadilla-del-Valle M., Riska P. F., Bardarov S. Jr, Small P. M., Ponce-de-Leon A., Jacobs W. R. Jr, Hatfull G. F., Sifuentes-Osornio J. 2003; Rapid identification and susceptibility testing of Mycobacterium tuberculosis from MGIT cultures with luciferase reporter mycobacteriophages. J Med Microbiol 52:557–561 [CrossRef]
     [Google Scholar]
  3. Bownds S. E., Kurzynski T. A., Norden M. A., Dufek J. L., Schell R. F. 1996; Rapid susceptibility testing for nontuberculosis mycobacteria using flow cytometry. J Clin Microbiol 34:1386–1390
     [Google Scholar]
  4. Boye E., Steen H. B., Skarstad K. 1983; Flow cytometry of bacteria: a promising tool in experimental and clinical microbiology. J Gen Microbiol 129:973–980
     [Google Scholar]
  5. Canetti G., Frosman S., Grosset J. H., Hauduroy P., Langerova M., Mahler H. T., Meissner G., Mitchison D. A., Sula L. 1963; Mycobacteria : laboratory methods for testing drug sensitivity and resistance. Bull WHO 29:565–578
     [Google Scholar]
  6. Cohn D. L., Bustreo F., Raviglione M. C. 1997; Drug-resistant tuberculosis: review of the worldwide situation and the WHO/IUATLD global surveillance project. Clin Infect Dis 24 (Suppl. 1):S121–S130 [CrossRef]
     [Google Scholar]
  7. Ibrahim P., Whiteley A. S., Barer M. R. 1997; SYTO 16 labelling and flow cytometry of Mycobacterium avium . Lett Appl Microbiol 25:437–441 [CrossRef]
     [Google Scholar]
  8. Khisimuzi M., Sherman D. R., Hickey M. J., Kreiswirth B. N., Morris S. W., Stover C. K., Barry C. E. III 1996; Biochemical and genetic data suggest that InhA is not the primary target for activated isoniazid-resistant clinical isolates of Mycobacterium tuberculosis . J Infect Dis 174:1085–1090 [CrossRef]
     [Google Scholar]
  9. Kirk S. M., Schell R. F., Moore A. V., Callister S. M., Mazurek G. H. 1998; Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid, and rifampin in 24 hours. J Clin Microbiol 36:1568–1573
     [Google Scholar]
  10. Moore A. V., Kirk S. M., Callister S. M., Mazurek G. H., Schell R. F. 1999; Safe determination of susceptibility of Mycobacterium tuberculosis to antimycobacterial agents by flow cytometry. J Clin Microbiol 37:479–483
     [Google Scholar]
  11. Norden M. A., Kurzynski T. A., Bowns S. E., Callister S. M., Schell R. F. 1995; Rapid susceptibility testing of Mycobacterium tuberculosis (H37Ra) by flow cytometry. J Clin Microbiol 33:1231–1237
     [Google Scholar]
  12. Pablos-Mendez A., Ravioglione M. C., Laszlo A. & 8 other authors; 1998; Global surveillance for antituberculosis-drug resistance, 1994-1997.World Health Organization-International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance. N Engl J Med 338:1641–1649 [CrossRef]
     [Google Scholar]
  13. Pina-Vaz C., Rodrigues A., Sansonetty F., Martinez-de-Oliveira J., Fonseca A. F., Mårdh P.-A. 2000a; Antifungal activity of local anesthetics against Candida species. Infect Dis Obstet Gynecol 8:124–137
     [Google Scholar]
  14. Pina-Vaz C., Sansonetty F., Rodrigues A., Martinez-de-Oliveira J., Fonseca A. F., Mårdh P.-A. 2000b; Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species. J Med Microbiol 49:831–840
     [Google Scholar]
  15. Pina-Vaz C., Sansonetty F., Rodrigues A., Costa-de-Oliveira S., Martinez-de-Oliveira J., Fonseca A. F. 2001a; Cytometric approach for rapid evaluation of susceptibility of Candida strains to antifungals. Clin Microbiol Infect 7:609–618 [CrossRef]
     [Google Scholar]
  16. Pina-Vaz C., Sansonetty F., Rodrigues A., Costa-de-Oliveira S., Martinez-de-Oliveira J., Fonseca A. F. 2001b; Susceptibility to fluconazole of Candida clinical isolates determined by FUN-1 staining and flow cytometry or epifluorescence microscopy. J Med Microbiol 50:375–382
     [Google Scholar]
  17. Pina-Vaz C., Costa-de-Oliveira S., Rodrigues A. G., Salvador A. 2004a; Laser Scanning Cytometer : a novel method for the detection of Mycobacterium in clinical samples. J Clin Microbiol 42:906–908 [CrossRef]
     [Google Scholar]
  18. Pina-Vaz C., Rodrigues A. G., Pinto E., Costa-de-Oliveira S., Tavares C., Salgueiro L., Cavaleiro C., Gonçalves M. J., Martinez-de-Oliveira J. 2004b; Antifungal activity of Thymus oils and their major compounds. J Eur Acad Dermatol Venereol 18:73–78 [CrossRef]
     [Google Scholar]
  19. Raviglione M. C., Snider D. E. Jr, Kochi A. 1995; Global epidemiology of tuberculosis.Morbidity and mortality of a worldwide epidemic. JAMA (J Am Med Assoc) 273:220–226 [CrossRef]
     [Google Scholar]
  20. Roth B. L., Poot M., Yue S. T., Millard P. J. 1997; Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain. Appl Environ Microbiol 63:2421–2431
     [Google Scholar]
  21. Rüsch-Gerdes S., Domehl C., Nardi G., Gismondo M. R., Welscher H.-M., Pfyffer G. E. 1999; Multicenter evaluation of the mycobacteria growth indicator tube for susceptibility of Mycobacterium tuberculosis to first-line drugs. J Clin Microbiol 37:45–48
     [Google Scholar]
  22. Tenover F. C., Crawford J. T., Huebner R. E., Geiter L. J., Horsburgh C. R. Jr, Good R. C. 1993; The resurgence of tuberculosis: is your laboratory ready?. J Clin Microbiol 31:767–770
     [Google Scholar]
  23. Torres M. J., Criado A., Palomares J. C., Aznar J. 2000; Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis . J Clin Microbiol 38:3194–3199
     [Google Scholar]
  24. World Health Organization 1997; Anti-tuberculosis drug resistance in the world: the WHO/IUATLD global project on anti-tuberculosis drug resistance surveillance 1994–1997. Technical bulletin 97.229 Geneva: World Health Organization;
     [Google Scholar]
/content/journal/jmm/10.1099/jmm.0.45627-0
Loading
Safe susceptibility testing of Mycobacterium tuberculosis by flow cytometry with the fluorescent nucleic acid stain SYTO 16
J. Med. Microbiol. 54, 77 (2005); https://doi.org/10.1099/jmm.0.45627-0
/content/journal/jmm/10.1099/jmm.0.45627-0
/content/journal/jmm/10.1099/jmm.0.45627-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error